To provide the information necessary for assessing risk and preventing tumorigenesis, the metabolism of N-acetylbenzidine and N,N/-diacetylbenzidine was assessed with rat liver microsomes from control and _-naphthoflavone-treated rats. Radio-labeled compounds were identified by co-elution with standard, unlabeled compounds, the structures of which were established unambiguously by mass spectrometry. The oxidation of 3H-N-acetylbenzidine to 3H-N'-hydroxy-N-acetylbenzidine (N'HA), 3H-N-hydroxy-N-acetylbenzidine (NHA), and 3H-ring oxidation products was assessed. For 3H-N,N/-diacetylbenzidine, the formation of 3H-N-hydroxy-N,N'-diacetylbenzidine (NHDA) and the 3H-ring oxidation product were assessed. With _-naphthoflavone-treated microsomes, the rate of NHA formation was 8-fold more than observed with control. While significant formation of ring oxidation products was demonstrated, the formation of N'HA was at the limit of detection. With control microsomes, N'HA was a maj or metabolite with more N'HA produced than NHA . While the oxidation of N,N/-diacetylbenzidine was not observed with control microsomes, significant formation of NHDA and ring oxidation product was observed with _-naphthoflavone-treated microsomes. Metabolism of 3H-N-acetylbenzidine and 3H-N,N/-diacetylbenzidine by _-naphthoflavone-treated microsomes was completely inhibited by the specific cytochrome P450 1A1/1A2 inhibitors (-naphthoflavone and ellipticine at 10 (M. Additional results are consistent with cytochrome P450 1A1/1A2 playing the major role in N-acetylbenzidine and N,N/-diacetylbenzidine metabolism by liver microsomes from control and _-naphthoflavone-treated rats. The formation of N'HA by control, but not by _-naphthoflavone-treated rats, and its insensitivity to inhibition by cytochrome P450 1A1/1A2 inhibitors was unexpected.